Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur. During incompatible interactions, bacterial populations do not increase, and progressive symptoms do not occur.
The hypersensitive response (“HR”) is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Horsfall et al., eds., Plant Disease: An Advanced Treatise Vol. 5, pp. 201–224, New York, N.Y.: Academic Press (1980); Mount et al., eds., Phytopathogenic Prokaryotes, Vol. 2, pp. 149–177, New York, N.Y.: Academic Press (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (≧107 cells/ml) of a limited host-range pathogen like Pseudomonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum) (Klement, Nature 199:299–300 (1963); Klement et al., Phytopathology 54:474–477 (1963); Turner et al., Phytopathology 64:885–890 (1974); Mount et al., eds., Phytopathogenic Prokaryotes, Vol. 2., pp. 149–177, New York, N.Y.: Academic Press (1982)). The capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Mount et al., eds., Phytopathogenic Prokaryotes, Vol. 2., pp. 149–177, New York, N.Y.: Academic Press (1982), these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren et al., J. Bacteriol. 168:512–22 (1986); Willis et al., Mol. Plant-Microbe Interact. 4:132–138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis et al., Mol. Plant-Microbe Interact. 4:132–138 (1991); Dangl, ed., Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals-Molecular and Cellular Mechanisms, pp. 79–98, Berlin: Springer-Verlag (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem et al., Trends Microbiol. 1:175–180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion protein elicitors of the hypersensitive response (He et al., Cell 73:1255–1266 (1993); Wei et al., J. Bacteriol. 175:7958–7967 (1993); Arlat et al., EMBO J. 13:543–553 (1994)). Hypersensitive response elicitor proteins, designated harpins, are proteins found in phytopathogens containing a type III secretion system and are typically glycine-rich, acidic, cysteine-lacking, heat stable proteins (He et al., Cell 73: 1255–1266 (1993).
The first of these proteins was discovered in E. amylovora Ea321, a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei et al., Science 257:85–88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for E. amylovora to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit. The P. solanacearum GMI1000 PopA1 protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat et al., EMBO J. 13:543–53 (1994)). However, P. solanacearum popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among gram-negative plant pathogens.
Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced from various organisms, including: HrpW from Erwinia amylovora (Kim et al., J. Bacteriol. 180(19):5203–5210 (1998)); HrpN from Erwinia chrysanthemi (Bauer et al., MPMI 8(4): 484–91 (1995)); HrpN from Erwinia carotovora (Cui et al., MPMI 9(7): 565–73 (1996)); HrpN from Erwinia stewartii (Ahmad et al., 8th Int'l. Cong. Molec. Plant-Microb. Inter. Jul. 14–19, 1996 and Ahmad et al., Ann. Mtg. Am. Phytopath. Soc. Jul. 27–31, 1996); hreX from Xanthomonas campestris (U.S. Patent Application Publ. No. 20020066122 to Wei et al.); HrpZ from Pseudomonas syringae pv. syringae (He et al., Cell 73:1255–1266 (1993); WO 94/26782 to Cornell Research Foundation, Inc.); and HrpW from Pseudomonas syringae pv. tomato (Charkowski et al., J. Bacteriol. 180:5211–5217 (1998)).
In electron microscopy studies, both HrpW and HrpZ of Pseudomonas syringae are associated with the type III secretion system pilus (Jin et al., Science 294:2556–2558 (2001); Jin et al., Molecular Microbiology 40:1129–1139 (2001)), suggesting that harpins work with the pilus to facilitate protein delivery into the plant cell. A P. syringae strain containing chromosomal deletions of hrpZ and hrpW has a reduced ability to cause the HR on nonhost plants, but it retains normal virulence on tomato (Charkowski et al., J. Bacteriol. 180:5211–5217 (1998)). This phenotype indicates that it is likely that there are more harpins in the genome.
The present invention is a further advance in the effort to identify, clone, and sequence hypersensitive response elicitor proteins or polypeptides from plant pathogens.